3528 Background: Anti-EGFR therapy (tx) in RAS wild-type (wt) metastatic colorectal cancer (mCRC) induces resistance through acquired genomic alterations. We aimed to define such alterations in Nationwide Cancer Genome Screening Project tissue and blood specimens, using next generation sequencing (NGS). Methods: Tumor specimens in patients (pts) with RAS wt mCRC were obtained from SCRUM-Japan GI-SCREEN (tissue) and GOZILA (circulating tumor DNA [ctDNA] from blood). Genomic alterations were compared using the Oncomine Comprehensive Assay (SCRUM) and Guardant360 (GOZILA), before anti-EGFR tx and after progression. Results: 373 total actionable alterations were identified in 71 pts with available matched tissue and ctDNA; 255 (68%) were acquired after anti-EGFR tx progression. Frequently seen acquired oncogenic alterations included KRAS mutations (27%) and amplifications (amps) of EGFR (41%), CDK6 (24%), BRAF (20%), MYC (17%), MET (14%), PIK3CA (11%), FGFR1 (11%), and KRAS (10%). Fusions of RET, ALK, and FGFR3 were newly acquired in 1-4%. Acquired alterations co-arose in multiple pathways, including the cell cycle, PI3K-AKT, and MAPK, although 29% of pts had none. Acquired mutations were less frequently clonal versus primary mutations (p<0.0001), but clonal acquired mutations were seen in several oncogenes, including EGFR, KRAS, and PIK3CA. A subset of acquired KRAS, MET, CCND2, and EGFR amps had high (>7) adjusted plasma copy numbers (ApCN). Acquired ERBB2 amps were identified in 3 pts (4%) with a median ApCN of 4, one of whom (ApCN=4.2), treated with dual HER2 blockade, progressed after 2 cycles. Conclusions: Our integrated analysis revealed that anti-EGFR tx of pts with RAS WT mCRC led to acquired genomic alterations in multiple oncogenic pathways. Although most acquired alterations were subclonal, a subset of oncogenic alterations had relatively high clonality and ApCN, suggesting potential targets for overcoming acquired resistance to anti-EGFR tx. Early progression in a pt with an ApCN of 4.2 suggests low-level/subclonal acquired alterations may not be effective treatment targets.